Oleate metabolism in microsomes from developing leaves ofPisum sativum L.

Microsomes from young leaves of pea,Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA:lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties.Abbreviations FA unesterified fatty acid (s) - PC phosphatidylcholines - 18:1 oleoyl moieties - 18:2 lmoleoyl moieties Dedicated to Professor Helmut K. Mangold, Bundesanstalt für Fettforschung, Münster, on his 60th birthday     

Lipid metabolism in microsomal fraction from photosynthetic tissue. Effects of catalase and hydrogen peroxide on oleate desaturation.

On incubation of microsomal fraction from pea (Pisum sativum L.) leaves with ammonium [1-14C]oleate or [1-14C]oleoyl-CoA in the presence of ATP, CoA, Mg2+ and NADH, the major reactions observed were those catalysed by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. The reaction catalysed by oleoyl phosphatidylcholine desaturase was specifically inhibited by H2O2, and this inhibitory effect was overcome by catalase (EC 1.11.1.6).     

Photo-induced dimerization of anthracene phospholipids for the study of the lateral distribution of lipids in membranes

The biosynthesis of linoleic acid has been investigated, using oleoyl-CoA as a substrate, in microsomal preparations from young leaves of Pisum sativum. Oleoyl moieties from oleoyl-CoA were preferentially acylated to lysophosphatidylcholine by an acyltransferase to produce an oleoylglycerophosphocholine. Kinetic data are presented which argue for a direct desaturation of the oleoyl moieties of this oleoyl glycerophosphocholine to linoleoyl moieties. There was no evidence of a subsequent acyltransfer of linoleoyl moieties either to form thioesters or oxygen esters in other complex lipids. The kinetics were also consistent with a functional coupling of the lysophosphatidylcholine acyltransferase with the oleate desaturase. There was little exchange of the oleoyl glycerophosphocholine from the bulk membrane lipid with that newly synthesised by the lysophosphatidylcholine acyltransferase. Rather, the newly synthesised oleoylglycerophosphocholine seemed to be directly channelled to the vicinity of the desaturase. The results are discussed in the context of 'metabolite channelling'. The consequences for desaturase activity and its regulation are also examined.     

Solubilisation of oleoyl-CoA thioesterase,oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase: The oleate desaturase system of pea leaf microsomes

Membrane-bound enzymes involved in oleate metabolism in microsomes from pea (Pisum sativum L.) leaves were solubilised using detergents, such as n-octyl glucoside, Triton X-100, digitonin or cholate. The detergents were found to be inhibitory to oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Detergent removal by dialysis resulted in the restoration of activity of both the solubilised oleoyl-CoA thioesterase and oleoyl-CoA:phosphatidylcholine acyltransferase. The putative components of the oleoyl phosphatidylcholine desaturase system were also partially solubilised.     

Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed.

1. [14C]Oleoyl-CoA was metabolized rapidly and essentially completely by microsomal preparations from developing safflower (Carthamus tinctorius) cotyledons, and most of the [14C]oleate was incorporated into 3-sn-phosphatidylcholine. 2. In aerobic reaction mixtures containing NADH2 the [14C]oleate in 3-sn-phosphatidylcholine was converted into [14C]linoleate without any change in the specific radioactivity of the lipid. Over a 60 min incubation period the extent of conversion of [14C]oleoyl phosphatidylcholine into [14C]linoleoyl phosphatidylcholine was generally greater than 60%. The rate of desaturation of endogenous [14C]oleoyl phosphatidylcholine labelled from [14C]oleoyl-CoA was much greater that of exogenous [14C]dioleoyl phosphatidylcholine the specific radioactivity of the oleoyl moiety of the lipid remained constant, indicating that labelled and unlabelled oleate were desaturated at the same rate. On this assumption an initial rate of desaturation of about 15 nmol of oleate desaturated/min per mumol of 3-sn-phosphatidylcholine was estimated. 4. [14C]Oleate esterified at positions 1 and 2 of both endogenous and exogenous 3-sn-phosphatidylcholine was desaturated. 5. Attempts to demonstrate the presence of an oleoyl-CoA desaturase in safflower microsomal fractions by the appearance of linoleoyl-CoA in reaction mixtures were inconclusive.     

Substrate specificities of the enzymes of the oleate desaturase system from photosynthetic tissue.

In the microsomal fraction from young pea (Pisum sativum L.) leaves, the oleoyl moieties from oleoyl-CoA are principally transferred to the sn-2 position of phosphatidylcholine by oleoyl-CoA:1-acyl-lysophosphatidylcholine acyltransferase. The major product of this acyl transfer is 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine. The 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine is subsequently converted into 1-palmitoyl(stearoyl)-2-linoleoyl phosphatidylcholine by the oleate desaturase complex without equilibrating with the bulk membrane phosphatidylcholine pool. Hence, both the acyl transfer to phosphatidylcholine and the subsequent desaturation of oleoyl moieties occur on the sn-2 position of phosphatidylcholine, and there is also a functional coupling of the acyltransferase and oleate desaturase.     

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.     

The Biosynthesis of Erucic Acid in Developing Embryos of Brassica rapa

The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [¹⁴C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of ¹⁴C in the 4 elongation carbons to ¹⁴C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [¹⁴C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy]-propanoic acid increased the accumulation of [¹⁴C]18:1 and the parallel accumulation of [¹⁴C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.     

The delta 12-desaturase from the house cricket, Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate

A novel delta 12-desaturase from animals, which converts oleic acid (18:1n-9) to linoleic acid (18:2n-6), was characterized in the house cricket, Acheta domesticus. The delta 12-desaturase product, linoleic acid, was determined by silver nitrate thin-layer chromatography, radio-gas-liquid chromatography and radio-high-performance liquid chromatography with the latter being used for routine analyses. Enzyme activity was located in the microsomal fraction of whole insect homogenates. NADPH or NADH was required for activity, with NADPH being the more efficient electron donor. In short incubation times with oleoyl-CoA as substrate, the highest amount of product, linoleic acid, was found as linoleoyl-CoA. With longer incubation periods, most of the linoleic acid was recovered in the polar lipid fraction containing phospholipid. Preincubation of the microsomal preparation in the absence of NADPH, which allowed 90% of the oleoyl moiety to be transacylated into complex lipid, resulted in no detectable desaturation upon addition of NADPH. These data indicate that the oleic acid moiety used as substrate was in the form of a CoA derivative and not in the form of a phospholipid, as it is for the plant delta 12-desaturase. This is the first characterization of a delta 12-desaturase from an animal system and the first report of a delta 12-desaturase that uses oleoyl-CoA as substrate.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Nonsense-mediated mRNA degradation of CtFAD2-1 and development of a perfect molecular marker for olol mutation in high oleic safflower (Carthamus tinctorius L.)

There are two types of safflower oil, high oleic (HO) with 70–75 % oleic acid and high linoleic (HL) with about 70 % linoleic acid. The original HO trait in safflower, found in an introduction from India, is controlled by a partially recessive allele ol at a single locus (Knowles and Bill 1964). In the lipid biosynthesis pathway of developing safflower seeds, microsomal oleoyl phosphatidylcholine desaturase (FAD2) is largely responsible for the conversion of oleic acid to linoleic acid. In vitro microsomal assays indicated drastically reduced FAD2 enzyme activity in the HO genotype compared to conventional HL safflower. A previous study indicated that a single-nucleotide deletion was found in the coding region of CtFAD2-1 that causes premature termination of translation in the HO genotypes, and the expression of the mutant CtFAD2-1Δ was attenuated in the HO genotypes compared to conventional HL safflower (Guan et al. 2012). In this study, we hypothesise that down-regulation of CtFAD2-1 expression in the HO genotype may be explained by nonsense-mediated RNA decay (NMD). NMD phenomenon, indicated by gene-specific RNA degradation of defective CtFAD2-1Δ, was subsequently confirmed in Arabidopsis thaliana seed as well as in the transient expression system in Nicotiana benthamiana leaves. We have developed a perfect molecular marker corresponding to the olol mutation that can facilitate a rapid screening and early detection of genotypes carrying the olol mutation for use in marker-assisted selection for the management of the HO trait in safflower breeding programmes.     

Incorporation of fatty acids into phosphatidylcholine is reduced during storage of human erythrocytes: Evidence for distinct lysophosphatidylcholine acyltransferases

The incorporation of [1-¹⁴C]palmitic or [1-¹⁴C]oleic acid into phosphatidylcholine and the effect on blood group antigen expression were examined in human erythrocytes stored at 4°C for 0-3 weeks. Blood drawn into EDTA was obtained by venepuncture from healthy volunteers. A 50% suspension of washed erythrocytes was incubated in buffer containing [1-¹⁴C]fatty acid for up to 60 min at 37°C with moderate shaking. Phosphatidylcholine was extracted and analyzed for uptake of radiolabelled fatty acid and phospholipid phosphorus content. Incorporation of [1-¹⁴C]palmitic or [1-¹⁴C]oleic acid into phosphatidylcholine was reduced during storage. The mechanism for the reduction in radiolabelled fatty acid incorporation into phosphatidylcholine was a 64% (p < 0.05) reduction in membrane phospholipase A2 activity. Although human erythrocyte membranes isolated from freshly drawn blood are capable of reacylating lysophosphatidylcholine to phosphatidylcholine, with storage, a markedly different substrate preference between palmitoyl-Coenzyme A and oleoyl-Coenzyme A was observed. Lysophosphatidylcholine acyltransferase activity assayed with oleoyl-Coenzyme A was unaltered with storage. In contrast, lysophosphatidylcholine acyltransferase activity assayed with palmitoyl-Coenzyme A was elevated 5.5-fold (p < 0.05). Despite these changes, storage of erythrocytes for up to 3 weeks did not result in altered expression of the various blood group antigens investigated. We conclude that the incorporation of palmitate and oleate into phosphatidylcholine is dramatically reduced during storage of human erythrocytes. The observed differential in vitro substrate utilization suggests that distinct acyltransferases are involved in the acylation of lysophosphatidylcholine to phosphatidylcholine in human erythrocytes.     

In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings

Exogenous [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both ¹⁴C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [¹⁴C]-oleate and [1-¹⁴C]linoleate esterification and of [1-¹⁴C]oleate desaturation to [1-¹⁴C]linoleate. [1-¹⁴C]Linoleic acid was esterified more rapidly than [¹⁴C]oleic acid and its desaturation product, [1-¹⁴C]α-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.     

Studies of the delta 12 desaturase of Carthamus tinctorius L

The delta 12 desaturase of developing safflower seeds responsible for the conversion of an oleoyl moiety to the linoleoyl moiety of phospholipids was further characterized. The protein concentration of the microsomal preparation, the oleoyl-CoA concentration (the primary substrate), short incubation periods, and the addition of lysophospholipids must be controlled to obtain optimal desaturation. No evidence could be obtained to implicate cytochrome b5 as the intermediate electron carrier. Attempts to solubilize the desaturase with a variety of detergents and chaotropic reagents were not successful. Brief exposure of the microsomal preparation to trypsin resulted in rapid loss of activity. The overall evidence would suggest that the delta 12 desaturase requires a reductant (NADPH), a NADPH:electron carrier reductase, an electron carrier, a specific desaturase, and an acyltransferase with oleoyl-CoA as the substrate to acylate lysophospholipid to the active oleoyl phospholipids (presumably phosphatidylcholine or phosphatidylethanolamine). The complexity of this system suggests that purification of the components and a reassembling of the purified components will be difficult.     

Temperature regulation of oleate desaturase in sunflower (Helianthus annuus L.) seeds

The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-¹⁴C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10 C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.Abbreviations DAF days after flowering - ODS oleoyl phosphatidylcholine desaturase - PC phosphatidylcholine - PE phosphatidylethanolamine - TAG triacylglycerol - 181 oleic acid - 182 linoleic acid To whom correspondence should be addressedThanks are due to M.C. Ruiz for skillful technical assistance. This work was supported by a grant from Junta de Andalucia, Spain.     

Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid

The activation of H⁺-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H⁺-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H⁺-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides.     

The incorporation of oleic and linoleic acids and their desaturation products into the glycerolipids of maize leaves

[1-¹⁴C]Oleic and [1-¹⁴C]linoleic acids were rapidly desaturated when incubated with maize leaves from 8-day-old plants and the labeled fatty acids, and their desaturation products, were rapidly incorporated into glycerolipids. Oleic acid was desaturated to linoleate at the rate of 0.7 nmol/100 mg tissue/h and further desaturated to linolenate at about one-third this rate. The rates of linolenate formation were similar when either oleic acid or linoleic acid was the substrate although there was a 2-h lag period when oleic acid was substrate. When radioactive oleic, linoleic, and linolenic acids were substrates, phosphatidylcholine was the most extensively labeled glycerolipid followed by monogalactosyldiacylglycerol. The relative rates of incorporation of label into individual glycerolipids are consistent with a movement of labeled fatty acids from phosphatidylcholine to monogalactosyldiacylglycerol and then to diagalactosyldiacylglycerol. The rates of labeling of phosphatidylcholine oleate and of phosphatidylcholine linoleate are consistent with a precursor-product relationship in that there was a delayed accumulation of phosphatidylcholine linoleate relative to that of phosphatidylcholine oleate and phosphatidylcholine linoleate continued to accumulate while phosphatidylcholine oleate declined. Linoleate formed from oleate was widely distributed in glycerolipids but neither phosphatidylcholine linolenate nor linolenate-containing diacylglycerol was detected at short and intermediate incubation times when either oleic or linoleic acid was substrate. The kinetics of incorporation of linoleate and linolenate into monogalactosyldiacylglycerol suggest a transfer of linoleate from phosphatidylcholine. The initial rate of accumulation of labeled linolenate in monogalactosyldiacylglycerol was very similar to the rate of desaturation of linoleate and it is suggested that desaturation of linoleate occurs while associated with monogalactosyl-diacylglycerol.     

The role of lysophosphatidylcholine in lipid synthesis by developing sunflower (Helianthus annuus L.) seed microsomes

The incorporation of oleate from oleoyl-CoA into lipids by microsomes from developing sunflower (Helianthus annuus L.) seeds has been investigated. Oleate was incorporated mainly into position 2 of phosphatidylcholine or released as free fatty acid. The addition of exogenous 1-acyl-lysophosphatidylcholine increased the incorporation of oleate into position 2 of phosphatidylcholine and decreased the release of free oleate. In the absence of exogenous lysophosphatidylcholine, the incorporation of oleate into phosphatidylcholine was limited by the amount of endogenous acceptor present. DH-990, an inhibitor of acyl-CoA:lysophosphatidylcholine acyltransferase, almost completely inhibited the incorporation of oleate from oleoyl-CoA into phosphatidylcholine at a concentration of 2.5 mM. These results indicate that the incorporation of oleate from oleoyl-CoA into microsomal phosphatidylcholine occurs mainly by the acylation of a 1-acyl-lysophosphatidylcholine acceptor rather than by acyl exchange between oleoyl-CoA and phosphatidylcholine. While the incorporation of oleoyl-CoA was completed within 2 to 5 min, exogenous 1-acyl-lysophosphatidylcholine was incorporated into phosphatidylcholine for up to 30 min. Addition of oleoyl-CoA resulted in an increase in both the rate and magnitude of lysophosphatidylcholine incorporation, which could not be accounted for by a stoichiometric reaction between the two substrates. Evidence is provided that free CoA had an independent stimulatory effect on the incorporation of lysophosphatidylcholine. The implications of this finding are discussed.     

A possible cyclic AMP-mediated regulation of microsomal fatty acyl-CoA desaturation system in Tetrahymena microsomes

Profound alterations in the microsomal fatty acyl-CoA desaturase activities and cyclic AMP production of a unicellular eukaryote, Tetrahymena pyriformis NT-1, originally grown in the glucose-deficient medium, were observed, following the administration of glucose or beta-adrenergic agonists such as epinephrine and isoproterenol. There was a great increase of stearoyl-CoA (delta 8) desaturase activity coincident with a 2-fold decrease of oleoyl-CoA (delta 12) desaturase activity over the first 2 h after administration of these compounds. During this period of time, it was found that the production in vivo of labeled oleic acid from [14C]acetic or [3H]palmitic acid increases 2-fold and the formation in vivo of each labeled linoleic and gamma-linolenic acids drastically decreases. Glucose or beta-adrenergic agonists caused an increase of stearoyl-CoA-stimulated reoxidation rate of NADH-reduced cytochrome b5 but depressed oleoyl-CoA-stimulated reoxidation rate of b5, indicating that both desaturase activities are controlled by the respective terminal components of the desaturase system. A significant and reproducible increase of adenylate cyclase activity and a slight decrease of cyclic AMP phosphodiesterase activity were observed to occur within the first 2 h after the addition of these compounds, when cyclic AMP content in Tetrahymena cell rose by 3-4-fold. Propranolol, a beta-adrenergic blocker, abolished the effects of glucose or beta-adrenergic agonists on the activities of fatty acyl-CoA desaturases and the terminal components as well as cyclic AMP production of cells. These results suggest that glucose and beta-adrenergic agonists may modulate the microsomal fatty acyl-CoA desaturase system in Tetrahymena by acting through the increase of intracellular cyclic AMP content.